This invention relates to luminescent substances and their use in clinical chemistry. More specifically, the invention relates to new luminescent cyclic hydrazides useful as analytical tools in clinical chemistry, to a process and intermediates for preparing the cyclic hydrazides, to cyclic hydrazide complexes or conjugates for use as reagents for luminescent immunoassay (LIA's), and to methods and kits for performing the assays.
Luminescent compounds have been used extensively in clinical chemistry; see reviews by F. Gorus and E. Schram, Clin. Chem. 25, 512 (1979) and T. P. Whitehead et al., Clin. Chem., 25, 1531 (1979). They have been demonstrated to be effective analytical tools in general, and a particular area of interest is their potential use as labels or tags for immunoassays. Presently, the most commonly used immunoassay is the radioimmunoassay (RIA). However, the RIA suffers from disadvantages inherent with the use of radioisotopes, such as radiation hazard and the relatively short half-life of certain isotopes. Hence, as expressed in the noted two reviews, interest in the development of nonisotopic labels has been increasing and in particular more attention is being focused on the possible replacement of RIA by LIA.
Indeed, LIA procedures have recently been reported for steroids, M. Pazzagli et al., Clin. Chem. Acta, 115, 287 (1981), F. Kohen et al., J. Steroid Biochem., 19, 413 (1983) and J. De Boever et al., Clin. Chem., 30, 1637 (1984); for proteins, P. J. Cheng et al., J. Immunol. Methods, 48, 159 (1982) and G. J. Barnard et al., Clin. Chem., 30, 538 (1984); and for other biological compounds, H. R. Schroeder et al., J. Immunol. Methods, 25, 275 (1979). These reports demonstrated the practicality of LIA procedures. However, the trend toward increasingly sophisticated techniques and the demand for detecting even more minute traces of analytes has created a need for more sensitive assays.
Two factors play a major role in the sensitivity of an immunoassay: the affinity of antibodies for the tracer and the specific activity of the latter. In LIA, the specific activity of the tracer is light emission and the quantum yield of the light is usually the most important parameter affecting the sensitivity of the assay.
Thus, the search for improved luminescent tags has rekindled the interest in cyclic hydrazides exhibiting chemiluminescence. The first cyclic hydrazide reported to have this property was luminol(5-amino-2,3-dihydro-1,4-phthalazinedione), H. O. Albrecht, Z. Phys. Chem., 136, 321 (1928). Since that report, a number of investigations have been directed to determining the factors that influence light production in luminol and related cyclic hydrazides. (Hereinafter, the term `cyclic hydrazide` will be used to designate compounds having a 2,3-dihydro-1,4-phthalazinedione ring system.) Several of these factors were identified very early by Drew and his coworkers as substituent effects, see H. D. K. Drew and F. H. Pearman, J. Chem. Soc., 26 and 586 (1937) and H. D. K. Drew and R. F. Garwood, J. Chem. Soc., 1841 (1937). Subsequent investigations have confirmed and expanded Drew's observations, see R. B. Brundrett and E. H. White, J. Amer. Chem. Soc., 96, 7497 (1974), and H. R. Schroeder and F. M. Yeager, Anal. Chem., 50, 1114 (1978).
In summary, the previous investigations have shown that in cyclic hydrazides the unimpeded resonance of electron-donating groups with the phenyl portion of the ring system generally exerts a favorable influence on the luminescent process as does mild steric interaction of certain substituents with adjacent carboxyls at C-1 and C-4, and that substitution of the heterocyclic moiety completely inhibits the process.
So far, the search for cyclic hydrazide luminescent labels has been directed mainly to derivatives of isoluminol(6-amino-2,3-dihydro-1,4-phthalazinedione) rather than luminol. Ligandchemiluminescent labeled conjugates derived from isoluminol are more efficient than those derived from luminol, apparently because of the relatively less steric hindrance exerted on the C-4 carbonyl of the isoluminol ring system. H. R. Schroeder et al., Methods in Enzymology, 57, 424 (1978) prepared several luminescent compounds and found that 6-[N-(4-amino-butyl)-N-ethylamino]-2,3-dihydro-1,4-phthalazinedione (ABEI) functioned as an efficient chemiluminescent label. The use of this derivative in LIA has resulted in a procedure with a sensitivity comparable to that of RIA: see Pazzagli et al., Kohen et al., and De Boever et al., supra.
Notwithstanding the advances made to date, there still remains a niche in clinical chemistry for more efficient luminescent compounds and, in particular, a need for markers that would give LIA systems of greater sensitivity.
Accordingly, the present application discloses the preparation of new cyclic hydrazides with improved light emission characteristics, useful as analytical tools in clinical chemistry. Moreover, adaptation of these compounds for LIA provides valuable reagents and assays with outstanding sensitivity.
The cyclic hydrazides are isoluminol derivatives. They are distinguished from previously known isoluminol derivatives by having an alkyl substituent in a meta position to the amino substituent on the phenyl portion of the ring system; notwithstanding the existence of broad generic disclosures of a myriad of compounds ranging in the millions, see S. Ikenoue et al., U.S. Pat. No. 4,207,112, June 10, 1980; T. J. N. Carter et al., European Patent Application No. 83301030.9, published Sept. 7, 1983; T. P. Whitehead et al., European Patent Application No. 84300725.3, published Aug. 22, 1984; and A. K. Campbell et al., U.S. Pat. No. 4,478,817, Oct. 23, 1984.